This project focuses on ZMPSTE24, a fascinating integral membrane zinc metalloprotease important for human health and longevity. ZMPSTE24 plays a critical role in the proteolytic processing of the farnesylated CAAX protein prelamin A, precursor of the nuclear scaffold component lamin A. Mutations in the genes encoding either prelamin A or ZMPSTE24 that impede prelamin A cleavage cause the devastating premature aging disorder Hutchinson-Gilford Progeria syndrome (HGPS) and a set of related progeroid diseases, due to the presence of the uncleaved and persistently farnesylated form of prelamin A. Notably, growing evidence suggests that diminished ZMPSTE24 processing of prelamin A is also a factor in normal physiological aging. My laboratory pioneered the study of ZMPSTE24 in early cycles of this project. We were the first to report its discovery in yeast, where it is calle Ste24p, and demonstrate that it has two distinct proteolytic activities. We went on to show that mammalian ZMPSTE24 mediates prelamin A processing. In the current project period we defined several features of the prelamin A substrate that are important for ZMPSTE24 cleavage and we showed that mild ZMPSTE24 disease alleles retain low but residual biochemical function, using a humanized yeast system. Through our sustained body of work on ZMPSTE24, we have developed a full arsenal of assays and tools that will facilitate the studies proposed here. The present proposal has been galvanized by the recently published structure of human ZMPSTE24 (and that of the nearly superimposable yeast Ste24p) which reveal a novel and surprising structure, never heretofore seen. The seven transmembrane spans of ZMPSTE24 form a voluminous, enclosed, water-filled intramembrane chamber that is capped at both ends. Notably, the ZMPSTE24 metalloprotease domain faces the chamber interior, so that substrate access is restricted. Prelamin A must be threaded into the chamber through one of several side portals. Here we propose mechanistic studies of human ZMPSTE24 (Aim 1) and its substrate prelamin A (Aim 2) to define the residues that mediate substrate access and activity, to identify interactors, and to precisely define the step(s) at which ZMPSTE24 disease alleles malfunction. These studies will provide the framework to understand the basis of premature aging disorders and to ensure optimal functioning of ZMPSTE24 as we age. In Aim 1 we will determine how ZMPSTE24 mediates proteolysis of prelamin A inside of an intramembrane chamber, using structure-guided mutagenesis and photo affinity probes to identify residues important for substrate entry, catalysis, binding, and product release. In Aim 2, we will use mutagenesis to define key features of the prelamin A substrate required for cleavage by ZMPSTE24, to better understand enzyme function. We will also identify ZMPSTE24/Ste24p binding partners that could contribute to substrate delivery or regulation of proteolytic activity. Together, our studies will reveal fundamental mechanistic principles relevant to nuclear membrane protein biology, premature aging disorders, and normal aging.